2 min read programming

Bioinformatics tools

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  1. Tools for quality control and adapter trimming -

FastQC - Checking the quality of raw reads, requires little memory,

cutadapt - Trimming the adapter sequences, supports wider range of sequencing platforms compared to fastx toolkit

Fastx toolkit - Checking read quality, trimming adapter sequences

RNA-Seqc - RNA-SeQC is used after FASTQC, which allows users to investigate the yield, alignment and duplication rate, GC bias, rRNA contamination rate, exon vs intron region vs intragenic alignment, 3’5’ bias, coverage, computationally intensive

ngsplot - Finding overall coverage of raw RNA-seq reads from transcription start site to transcription end site

TrimGalore - wrapper of cutadapt and fastqc

  1. Tools for alignment -

Bowtie - Aligner, not splice aware

Bowtie2 - Aligner, not splice aware

Tophat - Aligner, splice aware, successor of bowtie (wraps around bowtie), splice detection

Tophat2 - Aligner, splice aware

HISAT (Hierarchical indexing for spliced alignment of Transcripts) - A fast aligner, splice aware, specific to RNA, does NOT do soft clipping

HISAT2 - A fast aligner, splice aware, specific to RNA, Soft clipping done

STAR (Spliced Transcripts Alignment to a Reference) - A fast aligner (similar to HISAT), splice aware

BWA (Burrow wheeler aligner) - DNA aligner

Trinity - De novo transcriptome assembly

BSMAP - tolerate conversion of c to t in bisulfite sequencing

Bismark - tolerate conversion of c to t in bisulfite sequencing

Cufflinks - denovo transcriptome assembly

  1. Misc -

SAMtools - conversion of sam to bam, bam to sorted bam, removal of duplicate reads

Bedtools - bedintersect,

Cuffmerge

picard - removal of duplicate reads, merging sam files

diffRepeats - quantify repeat element enrichment / differential analysis of repeat elements in the DNA, algning short reads to a database of repeat sequences

diffReps - differential enrichment of a histone mark/transcription factor

mirdeep - de novo prediction of miRNA from RNA-seq data

ngsplot - can accept large files, runs with small memory footprints

phantompeek

chippeekanno

region_analysis

GeneOverlap - used to find the overlap between 2 sets of genes

  1. Identification of splicing events -

rMATS - exon skipping, intron retention

MISO - Mixture of isoforms, intron retention, skipped exon, mutually exclusive and alternative 3' UTR, splicing ratio b/w isoforms of same gene, splicing ratio between same isoform from 2 different conditions, uses bayesian theorem to iteratively infer splicing ratio. Allows to infer splicing ratios, both among isoforms of the same gene and between 2 conditions for the same isoform. Helps to identify contribution of histone/tf in regulation of AS.

  1. Peak calling -

DANPOS

MACS

Homer

  1. Quantification of aligned reads -

Cufflinks - Transcriptome abundance, de-novo transcriptome assembly, based on maximum likelihood principle, groups transcripts with the same transcription start site

StringTie - Transcriptome abundance, exon level quantification

Sailfish - no need of aligner, direct quantification

  1. Differential expression analysis -

Cuffdiff - following cufflinks, generates high false positives

Cuffdiff2 - generates high false positives

edgeR - Based on bayesian methods, negative binomial testing

DEseq - Based on bayesian methods, local regression, generates high false negatives (highly conservative output)

Limma

Voom - models the mean variance relationship on log transformed read counts

htseq count

8) Differential chip-seq analysis -

diffReps - to compare differential enrichment of a histone mark/transcription factor b/w 2 conditions, takes into account biological variations between a group of samples

9) Remove batch or unwanted effects from RNA-seq datasets -

ComBat

BatchQC

10) Bioinformatics databases -

repbase - documents repetitive DNA sequences across eukaryotic species

miRbase

piRNAbank

lncRNAdb

snornabase

SILVA

Rfam

NONCODE

fRNAdb

11) Tool for sampling reads from a fastq file -

seqtk